SUMMARY Metazoan cell migration contributes to developmental and homeostatic processes, including formation of tissues and organs, wound healing, tissue renewal and immune responses. Alterations in cell migration lead to developmental defects, inflammation, persistent wounds and metastasis. Deciphering mechanisms of cell migration thus has broad significance for understanding both physiological and pathological processes. Considerable effort has led to deep understanding of some of the critical processes that contribute to cell migration, including protrusion of the leading edge and formation of adhesions. Other important aspects of cell migration have received scant attention and there is a gap in our understanding of their mechanism and contribution to cell migration. This proposal seeks to fill this knowledge gap for two such processes: nuclear positioning and integrin recycling. Both these processes are essential for cell migration, but mechanistic understanding of how they do so is lacking. This proposal will build on results previously obtained in two previous projects in the Gundersen laboratory supported by NIGMS. For nuclear positioning, we will examine how the linker of nucleoskeleton and cytoskeleton (LINC) complex is mechanically reinforced to resist the large forces necessary to move the nucleus and will use and develop new tension sensors to directly measure forces on the nucleus. We will determine how the LINC complex selects actin filament or microtubules for nuclear movement and the functional consequences of these interactions for different modes of cell migration. We will test new hypotheses that the nucleus functions as a ?tension resistor? for actin filaments and as a polarity factor for microtubule trafficking. For integrin recycling, we will test the overall hypothesis that recycled integrins travel in an active conformation and that this seeds new adhesion formation in a polarized manner near the leading edge. We will use new integrin probes and a new integrin recycling system to identify sites of integrin exocytosis and their relationship to newly formed adhesions. In migrating cells, we will determine whether recycled integrin derives from the cell rear and contributes to nascent adhesion formation near the leading edge. We will identify the microtubule machinery that we hypothesize is responsible for the polarized reformation of adhesions from recycled integrins and test the possibility that the recycled integrin plays a role in integrin signaling. Our proposed studies on these two processes will advance understanding of the basic mechanisms of cell migration and potential identify new targets for intervening in cases when cell migration goes awry. The proposed studies will also provide fertile ground for postdoctoral fellows and graduate students to advance their training and develop their own projects.